All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.
Lipids in general are the building blocks of life. They are used as building blocks of membranes, cells and tissues, as energy sources, either immediate or stored, as precursors to a variety of other bio-molecules, as well as biochemical signals. In at biochemical processes lipids have an important role.
Many lipids, and especially triglycerides, are consumed in the human nutrition on a daily basis. In most cases, these lipids are metabolized and used for energy storage, precursors for biosynthesis of other lipids or bio-molecules. Whatever the fate of the lipids in the metabolic pathways, during and after their consumption, they interact with other nutrients or their metabolic products.
In human milk, and in most infant formulas, about 50% of the dietary calories are supplied to newborns as fat. More than 98% of this milk fat is in the form of triglycerides, which contain saturated and unsaturated fatty acids esterified to glycerol.
Fatty acids in human milk fat have a highly specific positional distribution on the glycerol backbone. This specific configuration is known to have a major contribution to the efficiency of nutrient absorption.
Palmitic acid (C16:0) is the predominant saturated fatty acid, constituting 20-25% of the fatty acids in mature human milk. 70-75% of this fatty acid are esterified at the sn-2 position of the triglycerides. In contrast, palmitic acid present in vegetable oils, which are most commonly used in the manufacture of infant formulas, is esterified at the sn-1 and sn-3 positions, while the sn-2 position is predominantly occupied by unsaturated fatty acids.
Triglyceride Digestion by the Infant
The triglyceride digestive process of the neonate is complex. It is initiated by a gastric phase catalyzed by gastric or lingual lipase [Hamosh M. (1990) Nutrition; 6:421-8]. This initial lipolysis allows maximal activity of pancreatic colipase-dependent lipase during the intestinal phase of digestion. The pancreatic lipase system attacks the triglyceride with a high degree of positional specificity. Lipolysis occurs predominantly at the sn-1 and sn-3 positions, yielding two free fatty acids and a 2-monoglyceride [Mattson F H. & Beck L H. (1956) J. Biol. Chem.; 219:735-740]. Monoglycerides are well absorbed independent of their constituent fatty acid. In contrast, the absorption of free fatty acids varies greatly, depending on their chemical structure. Mono and polyunsaturated fatty acids are well absorbed, as are saturated fatty acids of 12 carbons or less in chain length. The coefficient of absorption of free long chain saturated fatty acids i.e. palmitic acid is relatively low [Jensen C, et al. (1988) Am. J. Clin. Nutr.; 43:745-51], due in part to a melting point above body temperature (˜63°) and the tendency of these fatty acids to form hydrated fatty acid soaps with minerals such as calcium or magnesium at the pH of the intestine [Small D M. (1991) Annu. Rev. Nutr.; 11:413-434].
Several studies have demonstrated the preferential absorption of palmitic acid when present at the triglyceride sn-2 position [Lien E L. et al. (1997) J. Ped. Gastr. Nutr.; 52(2):167-174; Carnielli V P. et al. (1995) Am. J. Clin. Nutr.; 61:1037-1042; Innis S M. et al. (1993) Am. J. Clin. Nutr.; 57:382-390; Filer L. J. et al. (1969) J. Nutr.; 99:293-8]. Studies comparing the palmitic acid absorption of human milk and formulas conclude that the absorption of palmitic acid is higher in human milk [Chappel J E. et al. (1986) J. Pediatr.; 108:439-447; Hanna F M. et al. (1970) Pediatr.; 45:216-224; Tommarelli R M, et al. (1968) J. Nutr.; 95:583-90]. The greater absorption of fat and calcium in breast-fed infants compared with those fed formula has been ascribed to two factors: the presence in breast milk of a lipolytic enzyme (the bile salt-stimulated lipase) and the relatively high proportion of palmitic acid at the sn-2 position of the triglyceride [Hernell O. et al. (1988) Periniatal Nutrition. New York: Academic Press.; 259-272; Wang C S. et al. (1983) J. Biol. Chem.; 258:9197-9202]. Higher palmitic acid absorption was obtained with formulas rich in palmitic acid esterified in the sn-2 position of the triglycerides, than with those containing palmitic acid predominantly esterified in the sn-1,3 positions [López-López A. et al. (2001) Early Hum. Dev.; 65:S83-S94].
A study comparing the absorption of fat and calcium by infants fed a formula containing a blend of palm olein and soy oil (high levels of palmitic acid at the sn-1,3 positions) and a formula containing a blend of soy oil and coconut oil (low levels of palmitic acid) showed that the mixture of palm olein and soy oil, although providing the proportion of palmitic and oleic acids similar to those of human milk fat, was less absorbed [Nelson S E. et al. (1996) Am. J. Clin. Nutr.; 64:291-296]. Another study showed that fat absorption in infants fed formula containing lard was reduced when the high proportion of sn-2 palmitin in lard was reduced to 33% by chemical randomization [Filer (1969) id ibid.].
The composition of monoglycerides absorbed from the intestinal lumen is important to the fatty acid distribution of circulating lipids because about 70% of the fatty acids absorbed as sn-2 monoglycerides are conserved in the original position during re-esterification to form triglycerides in the intestinal cells [Small (1991) id ibid.].
Studies in piglets provided evidence that palmitic acid, when absorbed from milk or formula with rearranged triglycerides as a sn-2 monoglyceride, is conserved through the process of triglyceride reassembly in the enterocyte and secretion in plasma lipoprotein triglycerides [Innis S M. et al. (1995) J. Nutr.; 125:73-81]. It has also been shown that the distribution of saturated fatty acids in human milk and infant formula is a determinant of the fatty acid distribution of infant plasma triglycerides and phospholipids [Innis S M. et al. (1994) Lipids.; 29:541-545].
During the first year of life an infant's birth weight triples and the length is increased by 50%. To meet the requirements of their rapidly expanding skeletal mass, growing infants require a bioavailable source of calcium. For formula-fed infants, availability of calcium depends on the composition of the formula [Ostrom K. M. et al. (2002) J. Am. Coll. Nutr.; 21(6):564-569].
As mentioned above, the digestion of triglycerides involves lipolysis at the sn-1 and 3 positions and formation of free fatty acids and 2-monoglycerides. When palmitic acid is located at the sn-1,3 positions, as is the case in most infant formulas, it is released as free fatty acid which tends to form insoluble calcium soaps. In contrast, palmitic acid esterified to the sn-2 position, as in human milk, is unavailable to form calcium soaps [Small (1991) id ibid.].
Several studies have shown a correlation between formulas containing high levels of palmitic acid situated at the sn-1,3 positions of the triglyceride and reduction in calcium absorption [Nelson S E. et al. (1998) J. Amer. Coll. Nutr.; 17:327-332; Lucas A. et al. (1997) Arch. Dis. Child.; 77:F178-F187; Carnielli V P. et al. (1996) J. Pediatr. Gastroenterol. Nutr. 23:553-560; Ostrom (2002) id ibid.; Hanna (1970) id ibid.]. In addition, it was shown that dietary triglycerides containing palmitic acid predominantly at the sn-2 position, as in human milk, have significant beneficial effects on the intestinal absorption of fat and calcium in healthy term infants as well as in preterm infants [Carnielli (1996) id ibid.; Carnielli (1995) id ibid.; Lucas (1997) id ibid.]. Infants fed a formula containing high levels of palmitic acid at the sn-1,3 positions showed greater fecal excursion of calcium and, hence, a lower percentage absorption of calcium compared to infants fed a formula containing low levels of palmitic acid [Nelson (1996) id ibid.]. Fecal excretion of calcium was closely related to the fecal excretion of fat. This study also showed that urinary phosphorus excretion increased and phosphorus retention decreased when infants were fed the formula containing high levels of palmitic acid at the sn-1,3 positions. These findings presumably reflect lower availability of calcium for deposition in bones.
Another important issue which is associated with formula feeding is constipation in both term and preterm infants which, in the latter, can lead to life threatening complications. By contrast, constipation is rare in breast fed term infants. A study comparing breast fed and formula fed infant stool hardness and composition showed that calcium fatty acid soaps are positively correlated to stool hardness. Stools from formula-fed infants were significantly harder than those of the breast-fed infants suggesting different handling of saturated fatty acids [Quinlan P T. et al. (1995) J. Pediatr. Gastr. and Nutr.; 20:81-90].
In an attempt to overcome the decreased calcium absorption and hard stool phenomena, infant formula manufacturers tend to deviate from the fatty acid profile by replacing palmitic acid with lauric acid and, in some cases, by increasing the polyunsaturated fatty acid content. Studies have shown that fatty acid composition of the diet influences the fatty acid composition of developing infant tissue [Widdowson E. M. (1975) Br. Med. J.; 1:633-5; Carlson S E. et al. (1986) Am. J. Clin. Nutr.; 44:798-804; Innis S M. et al. (1990) Am. J. Clin. Nutr.; 5:994-1000; Koletzko B. et al. (1989) Eur. J. Pediatr.; 148:669-75] and thus the lipoprotein and lipid metabolism differ between breast-fed and formula-fed infants [Putnam J. C. et al. (1982) Am. J. Chin. Nutr.; 36:106-114; Innis S M. et al. (1992) Am. Coll. Nutr.; 11:63S-8S; Van Biervliet J P. et al. (1981) Acta. Paediatr. Scand.; 70:851-6].
Innis and colleagues [Innis (1993) id ibid.], when comparing three formulas containing similar amounts of saturated fatty acids—C8-C14, C16 from palm oil predominantly in the sn-1,3 positions), or C16 from synthesized triglyceride predominantly in the sn-2 position)—showed that the chain length of saturated fatty acids in infant formula influences the metabolism of the dietary oleic, linoleic and alpha-linolenic acids. This study also showed that the sn-2 configuration of C16 in human milk triglycerides seems to have unique properties that extend beyond absorption. These include effects on HDL and cholesterol concentrations, and the cholesterol ester fatty acid composition.
The impact of soap formation on calcium absorption can be significant. Many infant formulas contain sufficient saturated fatty acids to form soaps with virtually all the calcium available.
U.S. Pat. No. 4,876,107 (corresponding to EP 0 209 327) describes a substitute milk fat composition which is suitable as replacement fat in infant formulations. In this fat composition the total palmitic acid residues present is as high as 45%, with at least half of the fatty acid residues at the 2-position of the glycerol backbone being palmitic. The product has about 27% palmitic acid residues at the 1- and 3-positions, and the other substituents at the 1- and 3-positions are mainly unsaturated C16 and C18 fatty acid moieties. The fat composition is prepared by a specific process, in the presence of Hexane. Rather high levels of the fat compositions are required for the preparation of final infant formulations.
EP 0 496 456 also discloses substitute milk fat compositions. These compositions have a saturated fatty acid content at the sn-2 position of at least 40%, most of which are palmitic acid residues, and contain 0.2-7% linolenic acid moieties, 70% of which are bonded at the 1- and 3-positions of the glycerol moieties, the remaining acid moieties at the 1- and 3-positions, other than unsaturated fatty acids, are saturated C4-C12 fatty acids.
U.S. Pat. No. 5,658,768 discloses a multiple-step process for preparing triglyceride compositions in which more than 40% of the saturated fatty acid moieties are at the 2-position. Many of the steps involve enzymatic modifications.
In sum, one of the most pronounced differences between mother's milk and infant formulas is in the fat composition. In mother's milk, most of the saturated fatty acids (about 70%, mainly palmitic acid) are located at the sn-2 position of the triglycerides while the sn-1,3 positions are mainly occupied with unsaturated fatty acids. However, most infant formulas do not contain such composition and the result is the loss of energy (in the form of palmitic acid) and calcium by the infants. The reason for that is first and foremost, the limited availability of a fat mimicking the human breast milk fat. Currently, there is yet no natural alternative from a safe vegetal source. Limited sources are those of animal origin, which are extremely non-safe in a most delicate field like infant nutrition. One alternative in the past was to use lard, however health risks related to porcine viruses that can be transmitted to infants have caused this fat source to be eliminated. While there exist commercially available fats which mimic the fat composition of human breast milk, such as those described, e.g. in EP 0 209 327, they suffer several major drawbacks, inter alia the following:
Good blends are of very high cost and apparently limited availability, due to inferior methods of production. This is even more pronounced if the blends are to be used together with other new and relatively costly important nutrients, such as long-chain polyunsaturated fatty acid (LC-PUFA);
Commercial versions available on the market are inferior in terms of health benefits (only 43% of the total palmitic acid residues are esterified at the sn-2 position). A ratio of less than 50% (of the total palmitic acid is esterified at the sn-2 position) may have no meaningful benefits in terms of calcium and energy intake.
Production is by using a genetically modified enzyme, hence the product may be considered as GMO with the risks involved.
The products have to be incorporated to the formula blends at relatively high quantities, which may leave little room for any additional important oils and lipids to be incorporated without raising the total fat content of the formula.
Therefore, there are three important points when it comes to the triglyceride composition of human milk fat replacement:    1) The total amount of palmitic acid;    2) The ratio of palmitic acid at the sn-2 position (expressed as percent of palmitic acid at the sn-2 position from the total palmitic acid level);    3) The amount of oleic acid.
The amount of oleic acid is important in order to preserve the calcium and energy for the infant, and ensure normal and healthy development, since the fatty acids at the sn-1,3 positions of the oil component should be unsaturated. The higher the amount of unsaturated fatty acids, such as oleic acid, the better, since this indicates that most of the sn-1,3 positions are occupied by fatty acids that will not create harmful complexes with calcium. Consequently, the infant will not lose either energy (in the form of fatty acids) or calcium.
In order to find an optimal infant formula, wherein the amounts and composition of the fats are as close as possible to mother's milk, which would also be cost-effective, the present inventors have developed a new fat-based preparation in which the amount of palmitic acid residues at the sn-2 position of the triglycerides, and the amount of oleic acid are as close as possible to the optimum desired, as described below.
Thus, it is an object of the present invention to provide compositions typically comprising the fatty acids palmitic, oleic, linoleic and stearic acid, wherein up to 70% of the palmitic acid present is located in the sn-2 position. The invention also provides the process for preparation of said composition. Other uses and objects of the invention will become clear as the description proceeds.